Abstract The Notch signaling pathway is a highly conserved cell:cell communication pathway that plays critical roles in many aspects of metazoan development. Tight spatial and temporal regulation of this pathway is critical in many developmental decisions, and understanding the molecular mechanisms contributing to this elegant control is an area of broad interest. The proposed research focuses on a novel mechanism to regulate the pathway that requires direct ligand glycosylation by the fringe family of glycosyltransferases to modulate cis-interactions between Notch receptors and ligands. This model will be tested using mouse somitogenesis as a sensitive model to explore the fundamental importance of ligand glycosylation and cis-inhibition in Notch pathway regulation. Two aims will test the central hypothesis that ligand glycosylation by the fringe family of proteins modulates ligand interactions in cis and provides temporal regulation of the Notch pathway in the context of the segmentation clock that times vertebrate somitogenesis. First, cell culture analyses and mutagenesis will directly assess how ligand glycosylation affects protein interactions and ligand presentation in the Notch pathway, and biochemical approaches will examine how ligand glycosylation modulates protein:protein binding affinitites. In the second aim, a rigorous in vivo assessment of the function of ligand glycosylation will be completed. Completion of these aims will produce the first clear analysis of the functional relevance of ligand glycosylation as a locus of control for Notch signaling, and will integrate this model across scales from protein modification and trafficking in individual cells to cellular interactions and patterning in an organism. The work proposed here will provide the first rigorous analysis of the biological relevance of Notch ligand glycosylation by fringe proteins. Although the majority of Notch ligands contain conserved consensus sequences that would allow glycan addition by Pofut1 followed by glycan extension by fringe glycosyltransferases, the relevance of the modifications are unknown. Our work will exploit somitogenesis and the segmentation clock as a sensitive system that requires fringe glycosylation and cis-inhibition to rigorously test the biological significance of ligand glycosylation, examining the hypothesis that fringe modification of Notch ligands modulate the strength of protein interactions in cis, providing a novel mechanism to regulate the spatial and temporal activation of Notch signaling. We anticipate that the results from this work will have broad implications for our understanding of how the Notch pathway is regulated, allowing a pathway that appears straightforward on the surface to contribute to complex developmental decisions across metazoans.